Liposomes have been used as drug carriers for in vivo drug delivery. Liposome-encapsulated drugs have the following advantages:
the drugs are encapsulated within a relatively impermeable bilayer membrane where the drug is protected from the environment; PA1 liposomes can be taken up by cells without overt cytotoxic effects, thus enhancing the cellular uptake of the encapsulated material; PA1 encapsulation alters pharmacokinetics; and PA1 liposomes are natural, biodegradable and non-toxic. Mayhew et al., "Therapeutic Applications of Liposomes" in Liposomes, M. J. Ostro, ed. (Marcel Dekker, Inc., 1983) pp 289-341. It is believed that these same advantages will be observed when the liposomes encapsulate antigens.
There are several known process for making multilamellar liposome-encapsulated material on an industrial scale. Rao, "Preparation of Liposomes on the Industrial Scale: Problems and Perspectives," in LIPOSOME TECHNOLOGY, Vol. I, G. Gregordias, ed., (CRC Press, 1984) pp.247-257. In the most widely used of these, a thin lipid film (from an organic solvent) is deposited on the walls of a container, an aqueous solution of the material to be encapsulated is added, and the container is agitated. Bangham et al., J. Mol. Biol. 13: 238 (1965). Under the right conditions, this simple process results in the formation of multilamellar vesicles of liposomes trapping the material. Success of this procedure relies heavily on the formation of the thin lipid film, and variation in encapsulation is seen with different methods of agitation.
Belgian Patent No. 866697 describes an alternative method that does not rely on film formation. In this process, an organic solution of lipid is freeze-dried, resulting in a lyophilized product with physical properties that are conducive to easy hydration by an aqueous solution of the material to be encapsulated.
Problems associated with the known methods of production include variability between batches in the amount of material trapped within the liposomes (encapsulation efficiency), the volume of internal trapping space per amount (mg or .mu.mole) of lipid; the average diameter of individual liposomes, and size heterogeneity. Mayhew et al., supra. For example, Table I of Conrad et al., Biochim. Biophys. Acta 332: 36-46 (1974), shows that a standard deviation in encapsulation efficiency of 12-13% was found between 18 or 8 independently prepared liposome preparations. It is estimated that the range of values between individual preparations varied by about 50%. A low degree of encapsulation and high variation in encapsulation efficiency from batch to batch under otherwise similar conditions has been reported. Rao, supra.
Industrial methods effective at reducing variability in encapsulation efficiency have not heretofore been developed. The high variation in encapsulation efficiency has adverse consequences with respect to large-scale manufacture of liposome preparations for vaccine or drug delivery. Variation makes it virtually impossible to ensure uniformity from batch to batch, and makes quality control difficult.